Conformational Variants of the Individual HLA-I Antigens on Luminex Single Antigen Beads Used in Monitoring HLA Antibodies

Abstract

The strength of a donor-specific antibody should be assessed with a bead-specific mean fluorescence intensity cutoff based on TFL-006 reactivity against HLA-I beads, and HC-10 against iBeads, where the β2fHC or pepF-β2aHC normalized donor-specific antibody level would reveal the true anti-pepA-β2aHC reactivity associated with positive FCXM.

Characterization of the Anti-HLA Class I and II IgG Antibodies in Moroccan IVIg Using Regular Beads and Ibeads in Luminex Multiplex Single Antigen Immunoassay

“…Frequently, the native HLA may also exist, devoid of the peptide, as a HC with β2m. Indeed, using Flow cytometric Cross match analyses, with epitope specific monoclonal antibodies, confirmed the prevalence of peptide-associated β2m-associated HLA HC (pepA-β2aHC), on resting T-cells [43]. In addition to the above structural variants, a pool of β2m-free HLA as β2-free HC (β2fHC) was observed in proliferating human lymphoid cells [45], and in activated human T and B cells [46,47].…”

“…Percentage of increase refers to the same [43]. In other words, an MFI of an allele with iBeads that is lower than that of the regular bead would indicate that the affinity of the antibody is towards other HLA-I variants such as the β2m-free HC of HLA with or without peptides.…”

“…Whereas on iBeads, they may occur mostly as trimeric form HLA-I (peptide-associated heavy chain with β2m) or as dimeric form (peptide-free heavy chain with β2m) [43].…”

“…HLA-Ia alleles on regular Labscreen single antigen beads may occur as intact or native trimeric HLA (HLA heavy chain and β2m with peptide), as well as monomeric form such as heavy chain only, and dimeric form such as peptide-free heavy chain with β2m or β2m-free heavy chain with peptide [43]. Whereas on iBeads, they may occur mostly as trimeric form HLA-I (peptide-associated heavy chain with β2m) or as dimeric form (peptide-free heavy chain with β2m) [43].…”

“…The beads supplied by the manufacturer may have 2 categories of HLA proteins attached to the beads: HLA heavy-chain polypeptide only and heavy-chain polypeptides in association with Beta-2 microglobulin (β2m). Realizing the heterogeneity of proteins, the manufacturer recently developed iBeads (provided as Felix beads for in-house experimental use), in which regular HLA-Ia antigencoated microbeads are subjected to proprietary enzymatic treatment to remove or reduce the amount of heavy chains (also referred to as "denatured antigens" by the manufacturers) [42,43] . Briefly, the four lots of IVIg were titrated from 50 to 0.8mg/mL (diluted in 1X PBS, pH 7.2), and 20 µL of sample were incubated with 2µL of beads for 30 minutes at room temperature and on a shaker.…”